Cationic Liposomes Containing Disulfide Bonds in Delivery of Plasmid DNA

Abstract

Cationic liposomes are non-viral gene transfer vectors for in vitro and in vivo experiments. In the present studies, we investigated whether a disulfide linkage in a cationic lipid was reducible by cell lysate resulting in the release of plasmid DNA and enhanced gene transfection. We also investigated if the differences in transgene production were from differences in total amount of cellular associated plasmid DNA. We systematically compared the gene transfection of disulfide bond containing-cationic lipid, 1', 2'-dioleoyl-sn-glycero-3'-succinyl-2-hydroxyethyl disulfide ornithine conjugate (DOGSDSO), its non-disulfide-containing analog, 1', 2'-dioleyl-sn-glycero-3'-succinyl-1, 6-hexanediol ornithine conjugate (DOGSHDO), 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP). Two transgene reporter systems (i.e., luciferase and green fluorescent protein (GFP)) were used to address transgene transgene expression and transgene efficiency. Experiments with the luciferase expression plasmid resulted in transgene activity up to 11 times greater transgene production for the disulfide containing lipid in at least two different cell lines, COS 1 and CHO cells. When transgene expression was determined by GFP activity, DOGSDSO liposomes were four times greater than the non-disulfide lipid or positive control (DOTAP) liposomes. By quantifying nucleic acid uptake by flow cytometry it was also demonstrated that increase expression was not solely from an increase in cellular plasmid DNA accumulation. These results demonstrate that cationic lipids containing a disulfide linkage are a promising method for gene transfer.     

The location of the left-handedly curved DNA sequence affects exogenous DNA expression in vivo

The intranuclear disposition of a plasmid is extremely important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on plasmid expression were examined in vivo. A naked luciferase-plasmid was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. The location of the left-handedly curved sequence determined the transgene expression, without affecting the amount of intranuclear exogenous DNA. The plasmid containing the curved sequence at the location that results in the exposure of the TATA box out of the nucleosome core showed the highest expression. These results suggest that sequences with high histone affinity could control transgene expression from plasmids in vivo.     

CD1d induction in solid tumor cells by histone deacetylase inhibitors through inhibition of HDAC1/2 and activation of Sp1

CD1d is a MHC class-like molecule that presents glycolipids to natural killer T (NKT) cells, then regulates innate and adaptive immunity. The regulation of CD1d gene expression in solid tumors is still largely unknown. Gene expression can be epigenetically regulated by DNA methylation and histone acetylation. We found that histone deacetylase inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), induced CD1d gene expression in human (A549 and NCI-H292) and mouse (TC-1 and B16/F0) cancer cells. Simultaneous knockdown of HDAC1 and 2 induced CD1d gene expression. Sp1 inhibitor mitramycin A (MTM) blocked TSA- and SAHA-induced CD1d mRNA expression and Sp1 luciferase activity. Co-transfection of GAL4-Sp1 and Fc-luciferase reporters demonstrated that TSA and SAHA induced Sp1 luciferase reporter activity by enhancing Sp1 transactivation activity. The binding of Sp1 to CD1d promoter and histone H3 acetylation on Sp1 sites were increased by TSA and SAHA. These results indicate that TSA and SAHA could up-regulate CD1d expression in tumor cells through inhibition of HDAC1/2 and activation of Sp1.     

CD1d induction in solid tumor cells by histone deacetylase inhibitors through inhibition of HDAC1/2 and activation of Sp1

CD1d is a MHC class-like molecule that presents glycolipids to natural killer T (NKT) cells, then regulates innate and adaptive immunity. The regulation of CD1d gene expression in solid tumors is still largely unknown. Gene expression can be epigenetically regulated by DNA methylation and histone acetylation. We found that histone deacetylase inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), induced CD1d gene expression in human (A549 and NCI-H292) and mouse (TC-1 and B16/F0) cancer cells. Simultaneous knockdown of HDAC1 and 2 induced CD1d gene expression. Sp1 inhibitor mitramycin A (MTM) blocked TSA- and SAHA-induced CD1d mRNA expression and Sp1 luciferase activity. Co-transfection of GAL4-Sp1 and Fc-luciferase reporters demonstrated that TSA and SAHA induced Sp1 luciferase reporter activity by enhancing Sp1 transactivation activity. The binding of Sp1 to CD1d promoter and histone H3 acetylation on Sp1 sites were increased by TSA and SAHA. These results indicate that TSA and SAHA could up-regulate CD1d expression in tumor cells through inhibition of HDAC1/2 and activation of Sp1.     

Position effect variegation and epigenetic modification of a transgene in a pig model

Sequences proximal to transgene integration sites are able to regulate transgene expression, resulting in complex position effect variegation. Position effect variegation can cause differences in epigenetic modifications, such as DNA methylation and histone acetylation. However, it is not known which factor, position effect or epigenetic modification, plays a more important role in the regulation of transgene expression. We analyzed transgene expression patterns and epigenetic modifications of transgenic pigs expressing green fluorescent protein, driven by the cytomegalovirus (CMV) promoter. DNA hypermethylation and loss of acetylation of specific histone H3 and H4 lysines, except H4K16 acetylation in the CMV promoter, were consistent with a low level of transgene expression. Moreover, the degree of DNA methylation and histone H3/H4 acetylation in the promoter region depended on the integration site; consequently, position effect variegation caused variations in epigenetic modifications. The transgenic pig fibroblast cell lines were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine and/or histone deacetylase inhibitor trichostatin A. Transgene expression was promoted by reversing the DNA hypermethylation and histone hypoacetylation status. The differences in DNA methylation and histone acetylation in the CMV promoter region in these cell lines were not significant; however, significant differences in transgene expression were detected, demonstrating that variegation of transgene expression is affected by the integration site. We conclude that in this pig model, position effect variegation affects transgene expression.     

食品级乳酸乳球菌pNZ8149-luc质粒的构建及表达

目的:构建能够稳定表达萤火虫荧光素酶报告基因(luc)的乳酸乳球菌(Lactococcus lactis, L.lactis)食品级表达系统,以便后续研究对目的基因进行示踪。方法:从pGL4.10质粒中PCR扩增萤火虫荧光素酶报告基因,测序,克隆至载体pNZ8149,构建pNZ8149-luc表达质粒;电击转化宿主乳酸乳球菌NZ3900,采用乳糖筛选法获得重组的乳酸乳球菌,Nisin诱导,采用微孔板发光检测仪检测荧光素酶的存在,Western Blot检测目标蛋白luc的表达。结果:PCR扩增的荧光素酶报告基因成功克隆至pNZ8149质粒,并电击转化宿主乳酸乳球菌NZ3900,得到乳酸乳球菌表达系统NZ3900/pNZ8149-luc。Nisin诱导后,检测到荧光素酶随诱导时间的延长活性逐渐增强,时间超过24 h之后荧光素酶活性逐渐下降。Western Blot检测到目标蛋白luc在胞内表达。结论:成功构建了p NZ8149-luc表达载体,并能够在乳酸乳球菌体内稳定表达。     

Histone deacetylation directs DNA methylation in survivin gene silencing

DNA methylation and histone acetylation are major epigenetic modifications in gene silencing. In our previous research, we found that the methylated oligonucleotide (SurKex) complementary to a region of promoter of survivin could induce DNA methylation in a site-specific manner leading to survivin silencing. Here, we further studied the role of histone acetylation in survivin silencing and the relationship between histone acetylation and DNA methylation.First we observed the levels of histone H4 and H4K16 acetylation that were decreased after SurKex treatment by using the chromatin immunoprecipitation (ChIP) assay. Next, we investigated the roles of histone acetylation and DNA methylation in survivin silencing after blockade of histone deacetylation with Trichostatin A (TSA). We assessed survivin mRNA expression by RT-PCR, measured survivin promoter methylation by bisulfite sequencing and examined the level of histone acetylation by the ChIP assay. The results showed that histone deacetylation blocked by TSA reversed the effects of SurKex on inhibiting the expression of survivin mRNA, inducing a site-specific methylation on survivin promoter and decreasing the level of histone acetylation. Finally, we examined the role of histone acetylation in the expression of DNA methyltransferase 1 (DNMT1) mRNA. The results showed that histone deacetylation blocked by TSA reversed the increasing effect of histone deacetylation on the expression of survivin mRNA. This study suggests that histone deacetylation guides SurKex-induced DNA methylation in survivin silencing possibly through increasing the expression of DNMT1 mRNA.     

Protamine Sulfate Provides Enhanced and Reproducible Intravenous Gene Transfer by Cationic Liposome/DNA Complex

Abstract

A novel lipid/polycation/DNA (LPD) formulation has been developed for in vivo gene transfer. It involves the condensation of plasmid DNA with protamine sulfate, a cationic polypeptide, followed by the addition of DOTAP cationic liposomes. Compared with DOTAP/DNA complex, LPD offers greater protection of plasmid DNA against enzymatic digestion and gives consistently higher gene expression in mice via tail vein injection. The in vivo efficiency of LPD was dependent upon charge ratio and was also affected by the lipid used. Increasing the amount of DNA delivered induced an increase in gene expression. The optimal dose was approximately 50 μg per mouse, at which concentration approximately 10 ng luciferase protein per mg extracted tissue protein could be detected in the lung. Gene expression in the lung was detected as early as 1 h after injection, peaked at 6 h, and declined thereafter. Using LacZ as a reporter gene, it was shown that endothelial cells were the primary locus of transgene expression in both lung and spleen. No sign of inflammation in these organs was noticed. Since protamine sulfate has been proven to be non-toxic and only weakly immunogenic in humans, this novel vector may be useful for the clinical use of gene therapy.     

The nuclear localization sequence of the SV40 T antigen promotes transgene uptake and expression in zebrafish embryo nuclei

We report luciferase expression in zebrafish embryos after cytoplasmic injection of low copy numbers of plasmid DNA coupled to the SV40 T antigen nuclear localization sequence (NLS). Binding of NLS to plasmid DNA (pCMVL) occurs at room temperature in 0.25m KCl, as assayed by gel retardation at molar ratios of NLS:pCMVL of at least 100:1. Luciferase expression is induced in 35% of embryos with as low as 10³ NLS-bound pCMVL copies. With 10⁴ copies, the proportion of expression increases from 6% at 0:1 to 70% 100:1 NLS:pCMVL (p<0.01). The beneficial effect of NLS is abolished at DNA concentrations promoting high frequencies of transgene expression without NLS. Regardless of the DNA concentration, the use of NLS does not affect embryo viability for at least up to 10 days: The specificity of NLS on luciferase expression was tested by using a nuclear import deficient reverse NLS peptide (revNLS). revNLS binds to pCMVL, causing gel retardation similarly to NLS, but does not promote transgene expression. Binding of equimolar amounts of revNLS and NLS to DNA reduces by 50% the beneficial effect of NLS on transgene expression. The results suggest efficient targeting of NLS-bound plasmid DNA to the nucleus, and subsequent enhanced uptake of DNA by the nucleus. The data suggest that the use of NLS may reduce the need for using elevated DNA copy numbers in some gene transfer applications.     

FHL2转录激活结构域的定位

LIM蛋白家族成员FHL2 (fourandhalfLIMdomainprotein)在转录调节、细胞凋亡及肿瘤的发生发展中都起着重要作用。利用GAL4转录因子中的DNA结合结构域 (DBD)和含有与DBD结合序列的荧光素酶报告基因(GAL4 LUC)构建了哺乳动物细胞转录激活系统 ,并利用该系统定位了FHL2的转录激活结构域。首先将GAL4 DBD序列以正确读框插入到pcDNA3载体的多克隆位点中 ,构建成真核表达载体pDBD ,再将野生型FHL2及其不同片段以正确读框与pDBD中GAL4 DBD序列融合 ,构建成野生型FHL2及其缺失突变体表达载体。将这些表达载体分别瞬时转染 2 93T胚胎肾细胞 ,野生型FHL2及其缺失突变体都得到了表达。利用GAL4 荧光素酶报告基因对野生型FHL2及其不同突变体的转录激活活性检测表明 ,在 2 93T胚胎肾细胞和乳腺癌MCF 7细胞中 ,野生型FHL2具有转录激活活性 ,缺失N端半个LIM结构域使FHL2转录激活活性降低 ,缺失C末端第二个LIM结构域对FHL2的转录激活功能影响不大 ,缺失C末端最后一个LIM结构域则使FHL2的转录激活功能完全丧失 ,而C末端缺失 2个LIM结构域使FHL2转录激活活性又有所恢复。这说明FHL2C末端最后一个LIM结构域对其转录激活功能是必需的 ,而C末端第二个LIM结构域可能对FHL2的转录激活功能有负调控作用 ,这种负调控作用取决于     

Development of a Gateway-compatible two-component expression vector system for plants

Genetic transformation of plants offers the possibility of functional characterization of individual genes and the improvement of plant traits. Development of novel transformation vectors is essential to improve plant genetic transformation technologies for various applications. Here, we present the development of a Gateway-compatible two-component expression vector system for Agrobacterium-mediated plant transformation. The expression system contains two independent plasmid vector sets, the activator vector and the reporter vector, based on the concept of the GAL4/UAS trans-activation system. The activator vector expresses a modified GAL4 protein (GAL4-VP16) under the control of specific promoter. The GAL4-VP16 protein targets the UAS in the reporter vector and subsequently activates reporter gene expression. Both the activator and reporter vectors contain the Gateway recombination cassette, which can be rapidly and efficiently replaced by any specific promoter and reporter gene of interest, to facilitate gene cloning procedures. The efficiency of the activator–reporter expression system has been assessed using agroinfiltration mediated transient expression assay in Nicotiana benthamiana and stable transgenic expression in Arabidopsis thaliana. The reporter genes were highly expressed with precise tissue-specific and subcellular localization. This Gateway-compatible two-component expression vector system will be a useful tool for advancing plant gene engineering.

     

Sonoporation using microbubble BR14 promotes pDNA/siRNA transduction to murine heart

Naked plasmid DNA (pDNA) and short interfering RNA (siRNA) duplexes were transduced into adult murine heart by means of sonoporation using the third-generation microbubble, BR14. Plasmid DNAs carrying luciferase, beta-galactosidase (beta-gal), or enhanced green fluorescent protein (EGFP) reporter genes were mixed with BR14 and injected percutaneously into the left ventricular (LV) cavity of C57BL/6 mice while exposed to transthoracic ultrasound at 1MHz for 60s. Sonoporation at an output intensity of 2.0W/cm(2) and a 50% pulse duty ratio resulted in the highest luciferase expression in the heart. Histological examinations revealed significant expression of the beta-gal and EGFP reporters in the subendocardial myocardium of LV. Intraventricular co-injection of siRNA-GFP and BR14 with concomitant ultrasonic exposure resulted in substantial reduction in EGFP expression in the coronary artery in EGFP transgenic mice. The present method may be applicable to gain-of-function and loss-of-function genetic engineering in vivo of adult murine heart.     

Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system

Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Gene activation at a distance and telomeric silencing are not affected by yeast histone H1

Until recently, it was believed that the budding yeast Saccharomyces cerevisiae has no histone H1 gene. However, a search of the yeast genome database revealed a possible H1 homologue of 258 amino acids, termed yeast histone H1 (HHO1). The protein shows 36% identity to the human H1 core domain over a stretch of 93 amino acids. Unlike other H1 proteins, Hho1p has a second possible core domain which shows 43% identity to the first core domain. Since vertebrate H1 histone had been implied in gene repression as well as gene activation at a distance, we tested the effect of deleting the yeast H1-like gene on remote activation of a modified GAL1 promoter, which contains a synthetic GAL4 binding site close to the TATA box, and the natural UAS_G, consisting of four GAL4 binding sites. Different spacing up to 1.8 kb between the proximal binding site and the distal UAS_G enhancer revealed no differences in gene activation between wild-type and knockout strains. Overexpression of a heterologous histone H1 from sea urchin showed an overall inhibition of gene activation by the GAL1 promoter, whereas overexpression of the yeast histone H1 had no effect. Also, the expression of A1, ALPHA2 or SUC2 genes, all of which are known to be responsive to an altered chromatin structure, was unchanged in HHO1 knockout or HHO1-overexpressing strains when compared to wild-type cells. We also considered the possibility that HHO1 was involved in forming the heterochromatin at telomeres. On testing for telomeric silencing of a URA reporter gene introduced 1.3 kb away from the chromosomal end, we again observed no differences between wild-type and knockout strains. Thus, the yeast histone H1-like gene appears to have no role in gene activation at a distance or in silencing under the conditions tested. It remains to be seen whether the yeast H1 histone is a gene-specific regulator rather than a general chromatin-associated protein. Received: 16 April 1997 / Accepted: 4 July 1997     

Epigenetic Hierarchy within the MAGEA1 Cancer-Germline Gene: Promoter DNA Methylation Dictates Local Histone Modifications

Gene MAGEA1 belongs to a group of human germline-specific genes that rely on DNA methylation for repression in somatic tissues. Many of these genes, termed cancer-germline (CG) genes, become demethylated and activated in a wide variety of tumors, where they encode tumor-specific antigens. The process leading to DNA demethylation of CG genes in tumors remains unclear. Previous data suggested that histone acetylation might be involved. Here, we investigated the relative contribution of DNA methylation and histone acetylation in the epigenetic regulation of gene MAGEA1. We show that MAGEA1 DNA hypomethylation in expressing melanoma cells is indeed correlated with local increases in histone H3 acetylation (H3ac). However, when MAGEA1-negative cells were exposed to a histone deacetylase inhibitor (TSA), we observed only short-term activation of the gene and detected no demethylation of its promoter. As a more sensitive assay, we used a cell clone harboring a methylated MAGEA1/hph construct, which confers resistance to hygromycin upon stable re-activation. TSA induced only transient de-repression of the transgene, and did not lead to the emergence of hygromycin-resistant cells. In striking contrast, transient depletion of DNA-methyltransferase-1 in the reporter cell clone gave rise to a hygromycin-resistant population, in which the re-activated MAGEA1/hph transgene displayed not only marked DNA hypomethylation, but also significant reversal of histone marks, including gains in H3ac and H3K4me2, and losses of H3K9me2. Collectively, our results indicate that DNA methylation has a dominant role in the epigenetic hierarchy governing MAGEA1 expression.